Read Mapping Research Articles

First report of citrus-associated rhabdovirus in Australia in citrus.

Citrus-associated rhabdovirus (CiaRV), putatively classified in the Cytorhabdovirus genus as a strain of Cytorhabdovirus caricae, was first reported in China in 2021. Budwood from an asymptomatic kumquat (Citrus japonica Thunb.) collected in the Northern Territory, Australia in November 2021 a daughter tree was propagated from this budwood onto Carrizo citrange (Citrus ×insitorum Mabb.) rootstock and grown in a controlled environment greenhouse at the Elizabeth Macarthur Agricultural Institute (EMAI). Green bark tissue (100 mg) was sampled from the daughter plant and total RNA was extracted using Invitrogen Trizol TM Reagent (ThermoFisher Scientific, Australia), with DNA removed using DNaseI (Bioline, Australia). The Ramaciotti Centre for Genomics (Sydney, Australia) completed library preparation using a Truseq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, California, United States) and high throughput sequencing using an Illumina NovaSeq6000 sequencing system resulting in 128,234,594 raw reads (2x150bp). The data was trimmed using BBduk (Bushnell 2017) and de novo assembled using rnaSPAdes (v3.15.5) (Bushmanova et al. 2019). The resulting contigs were analysed using a local NCBI ref_viruses_rep_genomes BLAST database (downloaded 15 December 2023). There were eight contigs showing 95-99% identity to citrus leaf blotch virus (CLBV), family Betaflexiviridae (NC_003877), and three contigs (length - 6646, 6044, 1144 nucleotides; coverage - 2043, 174, 214x, respectively) generated showed 80.6, 78.6 and 83% identity to C. caricae (NC_055504). These contigs were analysed using the NCBI BLASTn database online and were 94.5% (MT302546), 90.7% (MT302546) and 94.7% (MT302545) identical to CiaRV isolates from China. Mapping of reads to MT302546 using Geneious Prime (Biomatters, New Zealand) resulted in a consensus sequence (PP998055) of a CiaRV genome with 92.8% identity to both MT302545 and MT302546 isolates. To confirm the presence of CiaRV in the sample, two primer sets, LF117/LR120 (Zhang et al. 2021) and CiaRV-F/CiaRV-R (Huang et al. 2023), were engaged to amplify regions of the L gene or nucleocapsid protein gene respectively. Bidirectional amplicon sequencing was followed by alignment using Geneious Prime (Biomatters) to the consensus sequence obtained by HTS, resulting in 100% identity. A further 176 citrus samples from Australia were tested by RT-PCR using the CiaRV-F/CiaRV-R primers that partially amplify the nucleoprotein (N) gene. Four trees (3 × kumquat, 1 × lemon (Citrus ×limon (L.) Osbeck)) were found to contain CiaRV including a kumquat which was held at EMAI as a living control for CLBV for over 20 years. Bidirectional Sanger sequencing was completed for each of the four positive samples with >99% similarity of each to the original sample. CiaRV was first detected in citrus (Citrus spp.), passionfruit (Passiflora edulis Sims) and paper bush (Edgeworthia chrysantha Lindl.) trees (Zhang et al. 2021) with no symptoms in infected citrus and foliar symptoms observed in the latter two hosts. The papaya virus E strain of C. caricae causes mild leaf vein clearing in Macroptilium lathyroides. When co-infected with cowpea severe mosaic virus (CPSMV), severe leaf mosaic is observed (Cornejo-Franco et al. 2022), noting that CPSMV causes mild leaf mottling in single infection of the host. The likely transmission of CiaRV by whitefly (Zhang et al. 2021), may result in the transmission of CiaRV to other hosts and possible mixed infections, increasing symptom severity. To our knowledge, this is the first report of CiaRV outside of China and it likely has been present in Australia for decades, possibly imported in budwood.

Refining dual RNA-seq mapping: sequential and combined approaches in host-parasitic plant dynamics.

Transcriptional profiling in host plant-parasitic plant interactions is challenging due to the tight interface between host and parasitic plants and the percentage of homologous sequences shared. Dual RNA-seq offers a solution by enabling in silico separation of mixed transcripts from the interface region. However, it has to deal with issues related to multiple mapping and cross-mapping of reads in host and parasite genomes, particularly as evolutionary divergence decreases. In this paper, we evaluated the feasibility of this technique by simulating interactions between parasitic and host plants and refining the mapping process. More specifically, we merged host plant with parasitic plant transcriptomes and compared two alignment approaches: sequential mapping of reads to the two separate reference genomes and combined mapping of reads to a single concatenated genome. We considered Cuscuta campestris as parasitic plant and two host plants of interest such as Arabidopsis thaliana and Solanum lycopersicum. Both tested approaches achieved a mapping rate of ~90%, with only about 1% of cross-mapping reads. This suggests the effectiveness of the method in accurately separating mixed transcripts in silico. The combined approach proved slightly more accurate and less time consuming than the sequential approach. The evolutionary distance between parasitic and host plants did not significantly impact the accuracy of read assignment to their respective genomes since enough polymorphisms were present to ensure reliable differentiation. This study demonstrates the reliability of dual RNA-seq for studying host-parasite interactions within the same taxonomic kingdom, paving the way for further research into the key genes involved in plant parasitism.

PangeBlocks: customized construction of pangenome graphs via maximal blocks

BackgroundThe construction of a pangenome graph is a fundamental task in pangenomics. A natural theoretical question is how to formalize the computational problem of building an optimal pangenome graph, making explicit the underlying optimization criterion and the set of feasible solutions. Current approaches build a pangenome graph with some heuristics, without assuming some explicit optimization criteria. Thus it is unclear how a specific optimization criterion affects the graph topology and downstream analysis, like read mapping and variant calling.ResultsIn this paper, by leveraging the notion of maximal block in a Multiple Sequence Alignment (MSA), we reframe the pangenome graph construction problem as an exact cover problem on blocks called Minimum Weighted Block Cover (MWBC). Then we propose an Integer Linear Programming (ILP) formulation for the MWBC problem that allows us to study the most natural objective functions for building a graph. We provide an implementation of the ILP approach for solving the MWBC and we evaluate it on SARS-CoV-2 complete genomes, showing how different objective functions lead to pangenome graphs that have different properties, hinting that the specific downstream task can drive the graph construction phase.ConclusionWe show that a customized construction of a pangenome graph based on selecting objective functions has a direct impact on the resulting graphs. In particular, our formalization of the MWBC problem, based on finding an optimal subset of blocks covering an MSA, paves the way to novel practical approaches to graph representations of an MSA where the user can guide the construction.

Benchmarking and building DNA binding affinity models using allele-specific and allele-agnostic transcription factor binding data

BackgroundTranscription factors (TFs) bind to DNA in a highly sequence-specific manner. This specificity manifests itself in vivo as differences in TF occupancy between the two alleles at heterozygous loci. Genome-scale assays such as ChIP-seq currently are limited in their power to detect allele-specific binding (ASB) both in terms of read coverage and representation of individual variants in the cell lines used. This makes prediction of allelic differences in TF binding from sequence alone desirable, provided that the reliability of such predictions can be quantitatively assessed.ResultsWe here propose methods for benchmarking sequence-to-affinity models for TF binding in terms of their ability to predict allelic imbalances in ChIP-seq counts. We use a likelihood function based on an over-dispersed binomial distribution to aggregate evidence for allelic preference across the genome without requiring statistical significance for individual variants. This allows us to systematically compare predictive performance when multiple binding models for the same TF are available. To facilitate the de novo inference of high-quality models from paired-end in vivo binding data such as ChIP-seq, ChIP-exo, and CUT&Tag without read mapping or peak calling, we introduce an extensible reimplementation of our biophysically interpretable machine learning framework named PyProBound. Explicitly accounting for assay-specific bias in DNA fragmentation rate when training on ChIP-seq yields improved TF binding models. Moreover, we show how PyProBound can leverage our threshold-free ASB likelihood function to perform de novo motif discovery using allele-specific ChIP-seq counts.ConclusionOur work provides new strategies for predicting the functional impact of non-coding variants.

Transriptome Analysis of Peripheral Blood Monocytes in Chronic Obstructive Pulmonary Disease Patients.

: The aim of the study was to perform monocyte transcriptome analysis for identification of differentially expressed genes and key disturbances in biological processes in these cells in COPD. . The study included three COPD patients and three smokers without bronchial obstruction. Monocytes were obtained from peripheral blood mononuclear cells using the plastic adhesion method. The cell purity achieved as a result of enrichment was approximately 90% according to flow cytometry data. The isolated RNA samples were purified from genomic DNA and ribosomal RNA. The samples were sequenced on a MGISEQ-200 sequencer in SE50 mode. Read mapping and transcript counting were performed in Salmon v1.10.1 software; further data processing was carried out in R software environment. : As a result of the analysis, 21 upregulated and 29 downregulated genes were found in monocytes from COPD patients. Among the genes with increased expression, the most significant were the noncoding RNAs PKD1P5-LOC105376752 and PKD1P4-NPIPA8, the role of which remains unclear, as well as SETDB2, RNASE6, SERPINE1, and MRC1. Downregulated genes, of which F8A2, ZDHHC19, CXCL9, CXCL10, HBA1, HBB, C2, CFB, CFD, MT1B, MT1G, and TIMP3 were of most interest, showed enrichment in seven gene ontology (GO) terms, including those related to response to lipopolysaccharides, hydrogen peroxide, copper ions, and complement activation. . The data obtained indicate inhibition of monocyte functional activity in COPD patients with a decrease in the ability to provide effective protection against microbial pathogens while weakening self-protection against reactive oxygen species. Upregulation of SERPINE1 and downregulation of TIMP3 may significantly contribute to airway remodeling and emphysema development in COPD.

TGIRT-seq of Inflammatory Breast Cancer Tumor and Blood Samples Reveals Widespread Enhanced Transcription Impacting RNA Splicing and Intronic RNAs in Plasma.

Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype but lacks unequivocal genomic differences or robust biomarkers that differentiate it from non-IBC. Here, Thermostable Group II intron Reverse Transcriptase RNA-sequencing (TGIRT-seq) revealed myriad differences in tumor samples, Peripheral Blood Mononuclear Cells (PBMCs), and plasma that distinguished IBC from non-IBC patients and healthy donors across all tested receptor-based subtypes. These included numerous differentially expressed protein-coding gene and non-coding RNAs in all three sample types, a granulocytic immune response in IBC PBMCs, and over-expression of antisense RNAs, suggesting wide-spread enhanced transcription in both IBC tumors and PBMCs. By using TGIRT-seq to quantitate Intron-exon Depth Ratios (IDRs) and mapping reads to both genome and transcriptome reference sequences, we developed methods for parallel analysis of transcriptional and post-transcriptional gene regulation. This analysis identified numerous differentially and non-differentially expressed protein-coding genes in IBC tumors and PBMCs with high IDRs, the latter reflecting rate-limiting RNA splicing that negatively impacts mRNA production. Mirroring gene expression differences in tumors and PBMCs, over-represented protein-coding gene RNAs in IBC patient plasma were largely intronic RNAs, while those in non-IBC patients and healthy donor plasma were largely mRNA fragments. Potential IBC biomarkers in plasma included T-cell receptor pre-mRNAs and intronic, LINE-1, and antisense RNAs. Our findings provide new insights into IBC and set the stage for monitoring disease progression and response to treatment by liquid biopsy. The methods developed for parallel transcriptional and post-transcriptional gene regulation analysis have potentially broad RNA-seq and clinical applications.

Identification of two effective sex-specific DNA markers in silver arowana (Osteoglossum bicirrhosum)

The silver arowana (Osteoglossum bicirrhosum), often referred to as a living fossil, presents significant challenges in sex identification based on morphological characteristics, with its sex determination mechanism remaining poorly understood. This issue poses considerable obstacles to its captive breeding efforts. Given the species' substantial value for both consumption and ornamental purposes, it is a critical candidate for aquaculture. Through genome sequencing, read mapping, and alignment analysis of five male and five female silver arowanas, along with one mixed pool of males, we successfully identified two sex-specific SNPs and confirmed a ZW model of sex determination in this ancient species. These two SNPs demonstrated remarkable accuracy, achieving 100 % success in genetic sex identification, as validated by Sanger sequencing of PCR products from 135 individuals. Both sex-specific SNPs are located approximately 26 kb upstream of the foxl2 gene, indicating a potentially pivotal role for the foxl2 gene in the sex-determination process of the silver arowana. Consequently, we have developed precise molecular markers for sex identification in the silver arowana, significantly enhancing reproductive efficiency and promoting further industrial development. This achievement lays a foundational basis for investigating the sex-determination mechanism of the silver arowana and other osteoglossid fish, thereby advancing aquaculture practices within this species.

Enhancing Variant Calling in Whole-Exome Sequencing Data Using Population-Matched Reference Genomes.

Whole-exome sequencing (WES) data are frequently used for cancer diagnosis and genome-wide association studies (GWAS), based on high-coverage read mapping, informative variant calling, and high-quality reference genomes. The center position of the currently used genome assembly, GRCh38, is now challenged by two newly published telomere-to-telomere (T2T) genomes, T2T-CHM13 and T2T-YAO, and it becomes urgent to have a comparative study to test population specificity using the three reference genomes based on real case WES data. Here we report our analysis along this line for 19 tumor samples collected from Chinese patients. The primary comparison of the exon regions among the three references reveals that the sequences in up to ∼ 1% target regions in T2T-YAO are widely diversified from GRCh38 and may lead to off-target in sequence capture. However, T2T-YAO still outperforms GRCh38 genomes by obtaining 7.41% more mapped reads. Due to more reliable read-mapping and closer phylogenetic relationship with the samples than GRCh38, T2T-YAO reduces half of variant calls of clinical significance which are mostly benign, while maintaining sensitivity in identifying pathogenic variants. T2T-YAO also outperforms T2T-CHM13 in reducing calls of Chinese-specific variants. Our findings highlight the critical need for employing population-specific reference genomes in genomic analysis to ensure accurate variant analysis and the significant benefits of tailoring these approaches to the unique genetic backgrounds of each ethnic group.

Abstract C093: Epigenetic insights and gene expression in African pan genome contig sequences

Abstract An often-overlooked component in our cancer genomics workflows is the reference genome. While most genomics analytical tools and findings are based on reference builds GRCh37 and GRCh38, these references are largely Eurocentric, and their representativeness among diverse populations has not been studied comprehensively. A previously published study conducted deep whole genome sequencing of over 900 individuals of African ancestry, where they revealed ∼296 million unique base pairs, assembled into 125,715 African Pan Genome (APG) contig sequences that are not represented in the reference genome. To further characterize these APG contig sequences, we sought to determine the epigenetic and expression potential of these sequences. We have previously reported preliminary efforts to characterize the unexplored epigenetic potential across the 125,715 APG contig sequences, where we have utilized Emboss CpG plot for CpG island prediction. Across all contig sequences, we identified 8,353 potential CpG islands across 6,352 contig sequences, where contigs had 1 to 9 predicted CpG islands. Taken together ∼5% of all APG contigs contained at least 1 predicted CpG island. To determine the expression potential of these contigs, we mapped RNAseq reads from an African ancestry-enriched triple negative breast cancer cohort to APG contigs that previously failed to map to the reference genome. Approximately 9% of APG contigs had transcriptomic reads mapping, and 1106 APG contigs had both reads mapping and predicted CpG islands. Given our RNAseq mapping from our African ancestry enriched cohort, we have begun to explore differential expression measures among contigs by African ancestry population groups, highlighting the potential for differential regulation at these understudied loci. Since the publication of the APG contig sequences, the recent telomere-to-telomere (T2T) reference has filled the gaps of GRCh37 and GRCh38, however, is even less diverse. With T2T containing novel sequences that were not present at the time the APG contigs were assembled, we performed BLASTn queries of APG contigs to the T2T genome, where preliminary analysis shows ∼17% fail to map to T2T. For those with BLASTn mapping results, we are currently assessing the coverage and identity of the mapping results to evaluate the alignment. In conclusion, these APG contig sequences harbor potential epigenetic regulatory function and expressed sequences not represented in our standard reference genomes. While some APG contig sequences are represented in the newer T2T genome, preliminary investigations show that these APG contig sequences map to the T2T genome in regions novel from GRCh37 and GRCh38. As we continue to explore these APG contig sequences, we may find ancestry-specific regulatory mechanisms not yet described. Citation Format: Rachel Martini, Kyriaki Founta, Sebastian Maurice, Jason White, Lisa Newman, Onyinye Balogun, Nyasha Chambwe, Melissa Davis. Epigenetic insights and gene expression in African pan genome contig sequences [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C093.

Cytoplasmic genomes of Jasminum sambac reveal divergent sub-mitogenomic conformations and a large nuclear chloroplast-derived insertion

BackgroundJasminum sambac, a widely recognized ornamental plant prized for its aromatic blossoms, exhibits three flora phenotypes: single-petal (“SP”), double-petal (“DP”), and multi-petal (“MP”). The lack of detailed characterization and comparison of J. sambac mitochondrial genomes (mitogenomes) hinders the exploration of the genetic and structural diversity underlying the varying floral phenotypes in jasmine accessions.ResultsHere, we de novo assembled three mitogenomes of typical phenotypes of J. sambac, “SP”, “DP”, and “MP-hutou” (“HT”), with PacBio reads and the “HT” chloroplast (cp) genome with Illumina reads, and verified them with read mapping and fluorescence in situ hybridization (FISH). The three mitogenomes present divergent sub-genomic conformations, with two, two, and four autonomous circular chromosomes ranging in size from 35.7 kb to 405.3 kb. Each mitogenome contained 58 unique genes. Ribosome binding sites with conserved AAGAAx/AxAAAG motifs were detected upstream of uncanonical start codons TTG, CTG and GTG. The three mitogenomes were similar in genomic content but divergent in structure. The structural variations were mainly attributed to recombination mediated by a large (~ 5 kb) forward repeat pair and several short repeats. The three jasmine cp. genomes showed a well-conserved structure, apart from a 19.9 kb inversion in “HT”. We identified a 14.3 kb “HT”-specific insertion on Chr7 of the “HT” nuclear genome, consisting of two 7 kb chloroplast-derived fragments with two intact ndhH and rps15 genes, further validated by polymerase chain reaction (PCR). The well-resolved phylogeny suggests faster mitogenome evolution in J. sambac compared to other Oleaceae species and outlines the mitogenome evolutionary trajectories within Lamiales. All evidence supports that “DP” and “HT” evolved from “SP”, with “HT” being the most recent derivative of “DP”.ConclusionThe comprehensive characterization of jasmine organelle genomes has added to our knowledge of the structural diversity and evolutionary trajectories behind varying jasmine traits, paving the way for in-depth exploration of mechanisms and targeted genetic research.

A reference genome for the Harpy Eagle reveals steady demographic decline and chromosomal rearrangements in the origin of Accipitriformes

The Harpy Eagle (Harpia harpyja) is an iconic species that inhabits forested landscapes in Neotropical regions, with decreasing population trends mainly due to habitat loss, and currently classified as vulnerable. Here, we report on a chromosome-scale genome assembly for a female individual combining long reads, optical mapping, and chromatin conformation capture reads. The final assembly spans 1.35 Gb, with N50scaffold equal to 58.1 Mb and BUSCO completeness of 99.7%. We built the first extensive transposable element (TE) library for the Accipitridae to date and identified 7,228 intact TEs. We found a burst of an unknown TE ~ 13–22 million years ago (MYA), coincident with the split of the Harpy Eagle from other Harpiinae eagles. We also report a burst of solo-LTRs and CR1 retrotransposons ~ 31–33 MYA, overlapping with the split of the ancestor to all Harpiinae from other Accipitridae subfamilies. Comparative genomics with other Accipitridae, the closely related Cathartidae and Galloanserae revealed major chromosome-level rearrangements at the basal Accipitriformes genome, in contrast to a conserved ancient genome architecture for the latter two groups. A historical demography reconstruction showed a rapid decline in effective population size over the last 20,000 years. This reference genome serves as a crucial resource for future conservation efforts towards the Harpy Eagle.

Personalized pangenome references.

Pangenomes reduce reference bias by representing genetic diversity better than a single reference sequence. Yet when comparing a sample to a pangenome, variants in the pangenome that are not part of the sample can be misleading, for example, causing false read mappings. These irrelevant variants are generally rarer in terms of allele frequency, and have previously been dealt with by filtering rare variants. However, this blunt heuristic both fails to remove some irrelevant variants and removes many relevant variants. We propose a new approach that imputes a personalized pangenome subgraph by sampling local haplotypes according to k-mer counts in the reads. We implement the approach in the vg toolkit ( https://github.com/vgteam/vg ) for the Giraffe short-read aligner and compare its accuracy to state-of-the-art methods using human pangenome graphs from the Human Pangenome Reference Consortium. This reduces small variant genotyping errors by four times relative to the Genome Analysis Toolkit and makes short-read structural variant genotyping of known variants competitive with long-read variant discovery methods.

Eye-Tracking in Formal Education. Understanding Map Reading Skills and Their Development

Eye-Tracking in Formal Education. Understanding Map Reading Skills and Their Development

Detecting differences between the map reading strategies of novice and expert students of geography: A pilot study

Detecting differences between the map reading strategies of novice and expert students of geography: A pilot study

A new graph-based metric for modeling aggregated gaze behavior differences during map reading

A new graph-based metric for modeling aggregated gaze behavior differences during map reading

A pilot study of adapting, validating and further developing a map skills model to improve the teaching and learning of map reading at primary and secondary school level

A pilot study of adapting, validating and further developing a map skills model to improve the teaching and learning of map reading at primary and secondary school level

Optimizing mobile map reading: An adaptive map approach in response to dynamic pedestrian context

Optimizing mobile map reading: An adaptive map approach in response to dynamic pedestrian context

Multi-omics approach for understanding the response of Bacteroides fragilis to carbapenems

BackgroundThe prevalence of Bacteroides fragilis isolates resistant to first-line beta-lactam drugs is increasing, resulting in reduced treatment efficacy. Investigating the bacterial transcriptome and proteome can uncover links between bacterial genes and resistance mechanisms. In this study, we experimentally assessed in vitro the transcriptional and proteomic profiles of B. fragilis exposed to SICs of meropenem, an effective antimicrobial agent, collected from patients with intra-abdominal diseases at Astana City Hospital, Kazakhstan. MethodsB. fragilis was cultured in brain heart infusion broth and sub-cultured every 48 h for 8 days in media with and without meropenem. Total RNA was extracted from the liquid cultures using a commercial RNeasy mini kit, and strand-specific RNA sequencing (RNA-seq) was performed on the DNBSEQ platform. Raw RNA-seq data were retrieved from BioProject No. PRJNA531645 and uploaded to the NCBI Sequence Read Archive (accession no. SRX22081155). Proteins of B. fragilis were extracted and separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by analysis of the eluted peptides using liquid chromatography–tandem mass spectrometry. Cluster analysis utilised the Database for Annotation, Visualisation, and Integrated Discovery. ResultsThe subinhibitory concentration (SIC) of meropenem was determined to be 0.5 μg/L (minimum inhibitory concentration: 1). Mapping of reads to the reference genome identified 2477 expressed genes in all B. fragilis BFR KZ01 samples. Ten differentially expressed genes (DEGs) were common across comparison groups during and post-antibiotic exposure (wMEM vs. MEM2 and MEM2 vs. rMEM8); however, no substantially enriched Gene Ontology terms were identified. The cluster analysis highlighted a significant enrichment cluster (W-0560 oxidoreductase) of DEGs following antibiotic withdrawal. In total, 859 B. fragilis proteins were identified, with the expressions of three proteins, 3-oxoacyl-[acyl carrier protein] reductase, acetyl-CoA carboxylase biotin carboxylase subunit, and beta-ketoacyl-ACP synthase III, being upregulated in the enriched protein folding category. Notably, chaperone proteins such as FKBP-type peptidyl-prolyl cis-trans isomerases (involved in the cis-trans isomerisation of prolyl peptide bonds) and GroES (a co-chaperone functioning with GroEL) were also identified. ConclusionsUnder the influence of low doses of antibiotics defense mechanisms are activated which contribute to the emergence of resistance. These results provide insight into the response of B. fragilis to meropenem exposure, mainly at the SIC, contributing to the understanding bacterial survival strategies under stress conditions.

Lightweight taxonomic profiling of long-read metagenomic datasets with Lemur and Magnet.

The advent of long-read sequencing of microbiomes necessitates the development of new taxonomic profilers tailored to long-read shotgun metagenomic datasets. Here, we introduce Lemur and Magnet, a pair of tools optimized for lightweight and accurate taxonomic profiling for long-read shotgun metagenomic datasets. Lemur is a marker-gene-based method that leverages an EM algorithm to reduce false positive calls while preserving true positives; Magnet is a whole-genome read mapping based method that provides detailed presence and absence calls for bacterial genomes. We demonstrate that Lemur and Magnet can run in minutes to hours on a laptop with 32 GB of RAM, even for large inputs, a crucial feature given the portability of long-read sequencing machines. Furthermore, the marker gene database used by Lemur is only 4 GB and contains information from over 300,000 RefSeq genomes. Lemur and Magnet are open-source and available at https://github.com/treangenlab/lemur and https://github.com/treangenlab/magnet.

Phenotypic and transcriptomics characterization uncovers genes underlying tuber yield traits and gene expression marker development in potato under aeroponics.

Transcriptome analysis in potato varieties revealed genes associated with tuber yield-related traits and developed gene expression markers. This study aimed to identify genes involved in high tuber yield and its component traits in test potato varieties (Kufri Frysona, Kufri Khyati, and Kufri Mohan) compared to control (Kufri Sutlej). The aeroponic evaluation showed significant differences in yield-related traits in the varieties. Total RNA sequencing was performed using tuber and leaf tissues on the Illumina platform. The high-quality reads (QV > 25) mapping with the reference potato genomes revealed statistically significant (P < 0.05) differentially expressed genes (DEGs) into two categories: up-regulated (> 2 Log2 fold change) and down-regulated (< -2 Log2 fold change). DEGs were characterized by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Collectively, we identified genes participating in sugar metabolism, stress response, transcription factors, phytohormones, kinase proteins, and other genes greatly affecting tuber yield and its related traits. A few selected genes were UDP-glucose glucosyltransferase, glutathion S-transferase, GDSL esterase/lipase, transcription factors (MYB, WRKY, bHLH63, and BURP), phytohormones (auxin-induced protein X10A, and GA20 oxidase), kinase proteins (Kunitz-type tuber invertase inhibitor, BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1) and laccase. Based on the selected 17 peptide sequences representing 13 genes, a phylogeny tree and motifs were analyzed. Real time-quantitative polymerase chain reaction (RT-qPCR) analysis was used to validate the RNA-seq results. RT-qPCR based gene expression markers were developed for the genes such as 101kDa heat shock protein, catechol oxidase B chloroplastic, cysteine protease inhibitor 1, Kunitz-type tuber invertase inhibitor, and laccase to identify high yielding potato genotypes. Thus, our study paved the path for potential genes associated with tuber yield traits in potato under aeroponics.